Sickle cell disease has the highest incidence for a population at risk of any genetically-derived disease. Three in every 1000 African Americans born have sickle cell anemia (SS disease). Vaso-occlusions from sickling of erythrocytes (RBCs) occur in most organs and are the initiating event in sickle cell retinopathy, which occurs in 15-30% of African Americans (depending on genotype) with sickle cell disease. We have identified three possible mechanisms of vaso-occlusion in the sickle cell retina in our prior studies with a rat model for sickle erythrocyte (RBC)-mediated vaso-occlusion: 1) tumor necrosis factor alpha (TNFa) stimulated adherence of sickle reticulocytes to vascular endothelium; 2) retention of dense sickled RBCs in hypoxic conditions; and 3) transient retention of sickled RBCs in normal rats as observed with the Rodenstock Scanning Laser Ophthalmoscope (SLO). Retention of sickled RBCs by Mechanism 1 was inhibited by antagonists of VLA-4, an integrin present on some sickled reticulocytes, and by antibodies against fibronectin administered intravenously. One of the proposed studies will determine the effect of TNFa on endothelial cells that stimulates reticulocyte adherence using parallel flow chambers, confocal microscopy, and scanning electron microscopy. Nitric oxide will be evaluated as a therapy for dense, irreversibly sickled cells mediated occlusions (Mechanism 2). The Proposal will also evaluate sites of sickled RBC-mediated vaso-occlusions for injury to endothelial cells, changes in the retinal milieu, and production of cytokines in the retina. Finally, all three mechanisms will be investigated in real time using the Rodenstock SLO. Therapies for prevention of vaso-occlusion or disruption of formed vaso-occlusions will be evaluated for their ability to shorten retention time or eliminate retention of sickled RBCs in real time using the SLO. In summary, this proposal will further investigate the mechanisms of vaso-occlusion in sickle cell retinopathy and will suggest strategies to prevent this initiating event in sickle cell retinopathy and necrosis in other organ systems.